Journal: bioRxiv

Article Title: Species-specific regulation of porcine STING stability and antiviral signaling via its K61 mediated K48 ubiquitination and proteasome degradation

doi: 10.64898/2026.03.26.714395

Figure Lengend Snippet: (A-B) HEK293T cells were co-transfected with mCherry-pSTING (20 ng) or its mutants together with pcGAS-HA (20 ng), plus ISRE-luc (10 ng) or NF-κΒ-luc (10 ng) and pRL-TK plasmid (0.4 ng) for 24 h. Luciferase activities were detected, and protein expressions were analyzed. (C-F) 3D4/21 cells in 24-well cell crawl slides were co-transfected with Flag-pSTING or K61R mutant together with KDEL-Red (C), mApple-SiT (D), MITO-GFP (E) or Lamp1-GFP (F) (each 0.5 μg) for 24 h, and then cells were fixed, stained and the colocalization of STING and its K61R mutant with organelles was examined by confocal fluorescence microscopy. (G) HEK293T cells in 6-well were transfected with Flag-pSTING or K61R mutant (1 μg/mL) for 24h, and stimulated with 2’3’-cGAMP (2 μg/mL) for 2 h. Then interaction between STING and TBK1/IRF3 was identified by Co-IP using anti-FLAG antibody, whereas rabbit IgG served as the control. (H-I) STING -/- 3D4/21 cells in 12-well were transfected with pmCherry-C1, mCherry-pSTING or K61R mutant (1 μg) for 24 h, and stimulated with 2’3’-cGAMP (2 μg/mL) for indicated times (H) or 6 h (I). Cells were harvested and analyzed by Western blotting (H) and RT-qPCR (I). (J-M) STING -/- 3D4/21 cells were transfected with Flag-pSTING or its mutants (1μg), and then stimulated with 2’3’-cGAMP (2 μg/mL) for 2 h, followed by infections with HSV-1 (MOI=0.01, 36 h) or PRV (MOI=0.01, 24 h). The viral titers in the supernatants from HSV-1 (J) or PRV (L) infected cells was measured by plaque assay, and the HSV-1 and PRV gD gene expressions were measured by RT-qPCR (K and M). ( N-P ) STING -/- MA104 cells were transfected and stimulated as in STING -/- 3D4/21 cells, followed by infections ASFV-GFP, infected cells were detected by fluorescence microscopy and cytometry, Western blotting and quantitative PCR. * p < 0.05 and ** p < 0.01.

Article Snippet: HEK293T cells (ATCC cat # CRL-3216), Vero cells (ATCC cat # CCL-81), Raw264.7 cells (ATCC cat # TIB-71), MDBK cells (ATCC cat # CCL-22) and MA104 cell (ATCC cat # CRL-2378.1) were cultured in DMEM (Hyclone Laboratories, USA) containing 10% fetal bovine serum (FBS, Vazyme Biotech Co., Ltd) and 100 IU/ml of penicillin plus 100 μg/ml streptomycin.

Techniques: Transfection, Plasmid Preparation, Luciferase, Mutagenesis, Staining, Fluorescence, Microscopy, Co-Immunoprecipitation Assay, Control, Western Blot, Quantitative RT-PCR, Infection, Plaque Assay, Cytometry, Real-time Polymerase Chain Reaction